63
Atherosclerosis, 25 (1976) 63-69 0 Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
ASCORBIC ACID METABOLISM DIET
IN RATS FED HIGH FAT CHOLESTEROL
B. NAMBISAN and P.A. KURUP Department
of Biochemistry,
University of Kerala, Trivandrum 695001
(India)
(Received 22nd January, 1976) (Accepted 11th March, 1976)
Summary The synthesis and catabolism of ascorbic acid has been studied in rats fed an atherogenic diet as also has the relation between the concentration of ascorbic acid and concentration of lipids in the tissues. The concentration of ascorbic acid was found to decrease in the serum, liver, spleen and adrenals, but not in the kidney, in the animals fed an atherogenic diet when compared with those fed normal diet. An inverse relationship was found between the concentration of ascorbic acid in the tissue and the concentration of lipids. The synthesis of ascorbic acid from D-glucuronolactone did not significantly differ in the liver and kidney, but decreased in the spleen in the animals fed the atherogenic diet. On the other hand, the catabolism of ascorbic acid significantly increased in the liver and spleen. Key words:
Adrenals - Aorta - Ascorbic acid- Atherogenic - Liver - Phospholipids -Spleen - Triglycerides
diet - Cholesterol - Kidney
Introduction
The effect of ascorbic acid on lipid metabolism in experimental animals fed an atherogenic diet has been investigated by several workers [l-17], but very few reports are available on the metabolism of ascorbic acid in animals fed an atherogenic diet. Ginter reported that guinea pigs fed a cholesterol diet and given ascorbic acid showed decreased tissue levels and increased urinary excretion of vitamin C, indicating that the vitamin requirement was increased by cholesterol [ 201. But rabbits and rats fed cholesterol showed increased tissue levels and increased urinary excretion of vitamin C, even though these animals, which can synthesize the vitamin had not been given vitamin C. Chevnyavskaya
64
observed that the ascorbic acid content of the liver in rabbits fed cholesterol was decreased in line with the degree of fat infiltration [ 181. Ginter et al. further reported that prolonged administration of a diet containing cholesterol to guinea pigs significantly stimulated the oxidation of ascorbic acid to COz [ 191. Apart from these, no other reports are available on the metabolism of ascorbic acid in cholesterol-fed animals. The synthesis of ascorbic acid and its catabolism have now been studied in rats (which can synthesize this vitamin) when fed a high fat cholesterol diet. Materials and Methods Male albino rats (Sprague-Dawley strain, average wt. 80 g) were divided into 2 groups of 15 rats each and fed as follows: Group I - fed normal diet Group II - fed high fat cholesterol diet The normal diet had the following composition (g/100 g): dextrin - 65, casein - 20, ground nut oil - 5, salt mixture - 4, vitamin mixture - 1, cellulose - 5. The salt mixture used had the following composition (mg/g): NaCl - 105.0, KC1 - 120, KH2P04 - 310, Ca,(P04)* - 149, CaC03 - 210, MnS04 (anhydrous) - 0.20, KzAlz(S04)4 - 24 Hz0 - 0.09, MgS04 (anhydrous) - 90.0, FeP04 * 4 Hz0 - 14.7, CuS04 - 5 Hz0 - 0.39, NaF - 0.57, KI - 0.05. The following trace elements were also added (mg/kg diet): ZnClz - 15.0 and COClz - 6 Hz0 - 0.15. The vitamin mixture contained (mg/lOO g diet): thiamine - 0.8, riboflavin 0.8, pyridoxine -0.6, niacin - 5.0, calcium pantothenate - 4.0, inositol20.0, choline chloride - 200, folic acid - 0.4, cyanocobalamin - 2.0 pg, biotin - 20 pg, vitamin A - 1000 IU, vitamin D - 150 IU, e-tocopherol - 12 mg and menadione - 0.3 mg. The high fat cholesterol diet contained (g/100 g): dextrin - 50, casein - 20, coconut oil - 15, cholesterol - 5, salt mixture - 4, vitamin mixture - 1, cellulose - 4.5, sodium cholate - 0.5. The vitamin mixture and salt mixture had the same composition as given above. The animals were maintained on the respective diets for a period of 3 months. They were then starved overnight, stunned by a blow at the back of the neck and killed by decapitation. The tissues were quickly removed and placed in ice-cold containers for the various estimations. Determination of total ascorbic acid in the tissues was carried out by the dinitrophenylhydrazine method of Roe and Kuether [ 211. Lipid levels of the tissues Total cholesterol was determined by the method of Schoenheimer and Sperry [ 221, phospholipids by the method of Zilversmit and Davis [ 231 and triglycerides by the method of Van Handel and Zilversmit [24], with the modification that a Florisil column was used to remove phospholipids. Preparation of liver, kidney and spleen homogenates The liver was homogenised in 4 vols./g cold iso-osmotic
(0.25
M) sucrose
65
solution at 5°C in an all glass PotterElvehjem homogeniser. The kidney spleen were homogenised in 3 vols./g 0.15 M KC1 in the same way.
and
System for the study of biosynthesis of L-ascorbic acid from D-glucoronolactone The system used was the same as described by Mukherjee et al [25] and contained sodium phosphate buffer pH 7.4 (20 mM), D-glucuronolactone (10 mM), tissue homogenate equivalent to approximately 100 mg of tissue, sodium pyrophosphate (50 n-&I) and KCN (50 m&f). The total volume was 2.5 ml and the mixture was incubated at 37°C for 90 min. The ascorbic acid synthesized was determined titrimetrically using 2,6-dichlorophenol-indophenol [ 261. System for the study of catabolism of L-ascorbic acid The system used was the same as that described by Mukherjee et al [25] and contained dehydroascorbic acid (10 pmols, freshly prepared by Brz oxidation of ascorbic acid), tissue homogenate (equivalent to 100 mg of tissue), glutathione (0.3 pmol), Tris-maleate buffer pH 6.8 (200 pmols) and magnesium chloride (20 pmols). The final volume was 3.0 ml. The system was incubated at 37” for 15 min. The reaction was stopped by addition of 1 ml of 20% w/v metaphosphoric acid containing 2% w/v of SnClz . The remaining dehydro-ascorbic acid was rapidly reduced with HZ S and filtered. Excess of HZ S was removed by bubbling Nz. A aliquot (0.1 ml) was taken for the estimation of 2.3 diketogulonic acid formed according to the method of Kajawa et ai. [27]. A zero time control was also run. Results (1) Concentration of ascorbic acid in the tissues The results are given in Table 1. The concentration of ascorbic acid in the serum, liver, spleen and adrenals significantly decreased in the animals fed the atherogenic diet when compared with those fed the normal diet. The decrease in the concentration of ascorbic acid in the kidney was not, however, significant. (2) Relation between the concentration of ascorbic acid and the concentration of lipids in the tissues The results are given in Table 1. Except in the kidney, the decreased concentration of ascorbic acid in the tissues was associated with a significant increase in total cholesterol, phospholipids and triglycerides. (3) Ascorbic acid synthesis in the liver, spleen and kidney The results are given in Table 2. The synthesis of ascorbic acid from D-glucuronolactone was not significantly affected in the liver and kidney, but decreased significantly. in the spleen. (4) Catabolism of ascorbic acid in the liver, spleen and kidney The results are given in Table 2.
TABLE 1
44.8 f 1.16
45.0 f 0.93
20.8 * 0.82
402.0 f 12.00
Liver
Spleen
Kidney
Adrenals
501 f 12.1
288.4 f 6.65
270.5 zk8.8
355.6 f 13.3
73.5 f 2.44
Triglyceride (mg glycerol/ 100 g tissue) f SEM 6.5 f 0.18 780 f 23.8 680 f 12.66 80.8 f 2.01 1123 * 2.95 ____
Phosphollpid (mg/lOO g tissue) * SEM 136.5 f 4.21 1313 i: 40.1 847.2 k 21.84 2450.1 f 49.02 1224.0 ?J31.2 __ ___
Average of the values from the individual tissues of 6 rats. Group I has been compared with Group II. aP < 0.01. b 0.01 < P < 0.05. In alI other cases P > 0.05 ’ mg/lOO ml of serum.
2.4 f 0.055
Ch&SteK31
(mg/lOO g tissue) + SEM
g
Ascorbic acid
I
ACID AND LIPIDS IN TISSUES
(mg/100 tia¶le) ? SEM
Group
OF ASCORBIC
Serum c
Tissue
CONCENTRATION
206.0 f 4.4 a ___
18.2 + 0.54
34.5 f 1.24 a
26.5 ?r 0.98 a
1.40 f 0.051 =
(mg/lOO g tie) t SEM
Ascorbic acid
Group II
879 f 20.4 a
a
2080 + 56.2 a
3520 f 116.5
a
1565 f 41.5 a
552.1 * 14.41 442 + 10.83
2885 f 59.3 a
876.2 f 20.8 a a
150.1 f 3.11 b
201.5 f 4.65 a
Phospholipid (mg/lOO g tissue) f SEM
Cholesterol (mg/lOO g tissue) +_SEM
311.0 ?r 8.1 a
89.9 f. 3.91
620 t 81.5 a
1050 f 21.5 a
11.8 + 0.43 a
(mg glycerol/ 100 g tissue) f SEM
Triglyceride
67 TABLE 2 ASCORBIC
ACID SYNTHESIS
AND DEGRADATION
CHOLESTEROL
DIET
Tissue
Ascorbic acid synthesized (rg/h/g + SEM -_
Liver Spleen Kidney
IN RATS
protein)
FED A NORMAL
AND A HIGH FAT
Diketogulonic acid (&h/g * SEM __
protein) _______
Group I
Group II
Group I
Group II
2250 + 45.6 352 i- 7.06 285 + 10.0
2260 + 66.7 281 + 8.05 a 260 * 5.50
48.8 f 0.98 96.4 f 3.70 41.4 f 1.42
66.8 i 1.96 a 273.6 + 5.92 a 38.0 ? 0.91
Average of the valued from the individual tissue of 6 rats. Group I has been compared with Group II. a P < 0.01. In all other cases P > 0.05.
The catabolism of ascorbic acid as measured by the diketogulonic acid formed was significantly increased in the liver and spleen. But there was no significant alteration in the kidney. Discussion The decreased concentration of ascorbic acid found in the tissues of rats fed an atherogenic diet agrees, except in the case of the kidney, with result8 reported by Chevnyavskaya [18] in rabbit8 fed cholesterol. It does, however, not agree with Ginter’s [20] findings in rabbit8 and rats, where increased tissue levels were found. The concentration of ascorbic acid in the tissues is inversely related to that of lipids, in agreement with Chevnyavskaya [ 181. No report8 are available about the synthesis and catabolism of ascorbic acid in rats fed a high fat cholesterol diet, except for a report [19] that prolonged administration of cholesterol to guinea pigs significantly stimulated the oxidation of ascorbic acid to co*. The results now obtained indicate that the synthesis of ascorbic acid from Dglucuronolactone did not significantly differ in liver and kidney, but decreased in the spleen of rats fed a high fat cholesterol diet. On the other hand, the catabolism of ascorbic acid significantly increased in the liver and spleen, but not in the kidney. Thus, the decreased concentration of ascorbic acid in the spleen in rats fed the high fat cholesterol diet might have been due to its decreased synthesis and increased catabolism, while in the liver it might have been due to increased catabolism, synthesis being unaffected. The fact that the synthesis and catabolism of ascorbic acid are not affected in the kidney is consistent with it8 unaltered concentration in the tissues. The pathway for the synthesis catabolism of ascorbic acid is given below: The enzymes concerned with the Synthesis from D-glucuronolactone are Dglucuronolactone reductase and L-gulonolactone oxidase. The decreased synthesis of ascorbic acid in the spleen of rat8 fed the high fat high cholesterol diet indicates that the activity of these enzymes may be decreased in the spleen, but unaffected in the liver and kidney. The enzyme in-
68 Dghmuronolactone Dglucuronolactone I! 11 II
uronolact + onase D&uronic acid
reductase
4 + L-gulonolactone IE II II + G~onic acid
Lgulonolactone
I
oxidase
1 2-keto-L-gulonolactone
I I
1f- spontaneous + Ascorbic acid 1 dehydroascorbic
1
acid
+ dehydroascorbatsse
2 Lxylonl~
: 3 dlketogulonic acid
acid
L-lyxonic ’ acid
volved in the degradation of dehydroascorbic acid to 2,3_diketogulonic acid is dehydroascorbitase. The increased concentration of 2,3-diketogulonic acid in the liver and spleen in the rats fed the high fat cholesterol diet indicates increased activity of this enzyme. Activity of this enzyme is, moreover, presumably unaltered in the kidney. References 1 Sitaramayya. C. and Ali. T.. Experimental hypercholesterolemia and atherosclerosis, Indian J. Physiol. Pharmacol.. 6 (1962) 192. 2 Abramron. M.A., Mechanism of ascorbic acid action in alimentary cholesterol atherosclerosis in rabbits, Materialy. Nauch. Konf. PO problem am Fuakts. Vzalmootnosheniya Mezdu Raslichen Sistemami Organirma Y Norme i Palos Ivanova Sb. (1962) 638. 3 Volkova, K.G.. Effect of ascorbic acid on total lipids in the aortic wall in experimental cholesterol atherosclerosis, Ateroskleroz. Sb.. (1961) 187. 4 Fernandez. M.A.. Gimeno, Lacuara. J.L., Gimeno, A.L., Lema. B. and Malinow, M.R.. Effect of ascorbic acid on experimental atherosclerosis in chickens, Acta Physiol. Latinoam. 10 (1960) 168. 5 Chang, S.C., Effect of mechanical trauma, cortisone and vitamin C upon atherosclerosis in cholesterol fed rats, New Engl. Med. J., 8(8) (1965) 79. 6 Sokoloff, B., Hori, M.. Sadhof, C.. McConnell, B. and Imai. T., Effect of ascorbic acid on certain fat metabolism factors in animals and man, J. Nutr.. 91 (1967) 107. 7 Rzakulleva. D.M.. Effect of ascorbic acid on the development of atherosclerosis, Izv. Akad. Nauk. Azerb. SSR. Ser Biol. Nauk. No. 2 (1969) 110. 8 Novitskil. A.A., Ivanov, A.I. and Reshetnev. V.G.. Changes of intensity of cholesterol biosynthesis under the effect of ascorbic acid at early stages of experimental atherosclerosis, Patol. Fiaiol. Eksp. Ter.. 13 (1969) 59. 9 Ginter. E., Ondreicka, R.. Bobek, P. and Simko. V.. Influence of chronic vit. C deficiency on fatty acid composition of blood serum, liver triglycerides and cholesterol esters in guinea pigs, J. Nutr. 99 (1969) 261. 10 Giiter. E., Babala, J. and Cerven, J.. Effect of chronic hypovitaminosis C on the metabolism of cholesterol and atherogenesls ln guinea pigs, J. Atheroscler. Res.. 10 (1969) 341. 11 Ginter. E. and Nemco, R.. Metabolism of [1-14C]acetate in guinea pids with chronic vitamin C hyposaturation, J. Atheroscl. Res., 10 (1969) 273. 12 Novitskii. A.A.. Correlation of the metabolism of cholesterol and ascorbic acid during prolonged hypercholesterolemea. Tr. Kuibyshev. Med. Inst., 56 (1969) 122. 13 Novitskii. A.A., Effect of ascorbic acid on the biosynthesis of cholesterol in the early stages of experimental atherosclerosis, Kardiologiya, 10 (1970) 118. 14 Ginter, E., Babala, J. and Polonyova, E., Vitamin C and lipid metabolism in rabbits fed an atherogenic diet, Biologia. 25 (1970) 579. 15 Glnter, E. and Babala, J., Effect of high intraperitoneal doses of vitamin C in guinea pigs fed atherogenie diet, Biologia. 26 (1971) 449. 16 Novitskli, A.A.. Influence of vitamin C on cholesterol netabolism in liver in experimental atherosclerosis, CoretVasa, 11 (1969) 302. 17 FWinami. T., Okado, K.. Senda. K., Sugimura, M. and Klshlkawa, M., Experimental atherosclerosis
69
18
with ascorbic acid deficiency, Jap. Circ. J., 35 (1971) 1559. Chemyavskaya. G.L., Histochemistry of ascorbic acid in the liver during
19
esterolemia in rabbits, Ginter. E. and Zloch,
20 21 22 23 24 25 26 27
Tr. volgograd. Gos. Med. Inst., 23 (1970) 131. 2.. Raised ascorbic acid consumption in cholesterol
experimental
hyperchol-
fed guinea pigs, Int. J. Vit.
Nutr. Res.. 42 (1972) 72. Ginter, E.. Effect of dietary cholesterol on vitamin C metabolism in laboratory animals. Acta Med. Acad. Sci. Hung.. 27 (1970) 23. Hawk, P.B., Oser. B.L. and Summerson, W.H., In: Hawk’s Physiological Chemistry, 14th edition, McGraw-Hill, New York. N.Y., 1965. p. 703. Schoenheimer, R. and Sperry. W.M.. In: Hawk’s Physiological Chemistry, 14th edition, McGraw-Hi& New York, N.Y.. 1965. p. 1062. Zilversmit, D.B. and Davis, A.K., Microdetermination of phospholipids. J. Lab. CIin. Med., 35 (1950) 155. Van Handel, E. and Wversmit, D.B.. Micromethod for direct determination of serum triglycerides. J. Lab. Clin. Med., 50 (1957) 152. Mukherjee, D.. Kar, N.C., SasmaI, N. and Chatterjee. G.C.. The influence of dietary protein on ascorbic acid metabolism in rats, Biochem. J.. 106 (1968) 627. GIick. D.. Methods of Biochemical Analysis. Vol. 1, Interscience, New York, N.Y., 1957, p. 120. Kagawa. Y.. Hisashi. T. and Shimazono, N. In: D. GIick (Ed.). Methods of Biochemical Analysis, Vol. 1. Interscience.
New York,
N.Y..
1957.
p. 132.
Atherosclerosis, 25 (1976) 63-69 0 Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
ASCORBIC ACID METABOLISM DIET
IN RATS FED HIGH FAT CHOLESTEROL
B. NAMBISAN and P.A. KURUP Department
of Biochemistry,
University of Kerala, Trivandrum 695001
(India)
(Received 22nd January, 1976) (Accepted 11th March, 1976)
Summary The synthesis and catabolism of ascorbic acid has been studied in rats fed an atherogenic diet as also has the relation between the concentration of ascorbic acid and concentration of lipids in the tissues. The concentration of ascorbic acid was found to decrease in the serum, liver, spleen and adrenals, but not in the kidney, in the animals fed an atherogenic diet when compared with those fed normal diet. An inverse relationship was found between the concentration of ascorbic acid in the tissue and the concentration of lipids. The synthesis of ascorbic acid from D-glucuronolactone did not significantly differ in the liver and kidney, but decreased in the spleen in the animals fed the atherogenic diet. On the other hand, the catabolism of ascorbic acid significantly increased in the liver and spleen. Key words:
Adrenals - Aorta - Ascorbic acid- Atherogenic - Liver - Phospholipids -Spleen - Triglycerides
diet - Cholesterol - Kidney
Introduction
The effect of ascorbic acid on lipid metabolism in experimental animals fed an atherogenic diet has been investigated by several workers [l-17], but very few reports are available on the metabolism of ascorbic acid in animals fed an atherogenic diet. Ginter reported that guinea pigs fed a cholesterol diet and given ascorbic acid showed decreased tissue levels and increased urinary excretion of vitamin C, indicating that the vitamin requirement was increased by cholesterol [ 201. But rabbits and rats fed cholesterol showed increased tissue levels and increased urinary excretion of vitamin C, even though these animals, which can synthesize the vitamin had not been given vitamin C. Chevnyavskaya
64
observed that the ascorbic acid content of the liver in rabbits fed cholesterol was decreased in line with the degree of fat infiltration [ 181. Ginter et al. further reported that prolonged administration of a diet containing cholesterol to guinea pigs significantly stimulated the oxidation of ascorbic acid to COz [ 191. Apart from these, no other reports are available on the metabolism of ascorbic acid in cholesterol-fed animals. The synthesis of ascorbic acid and its catabolism have now been studied in rats (which can synthesize this vitamin) when fed a high fat cholesterol diet. Materials and Methods Male albino rats (Sprague-Dawley strain, average wt. 80 g) were divided into 2 groups of 15 rats each and fed as follows: Group I - fed normal diet Group II - fed high fat cholesterol diet The normal diet had the following composition (g/100 g): dextrin - 65, casein - 20, ground nut oil - 5, salt mixture - 4, vitamin mixture - 1, cellulose - 5. The salt mixture used had the following composition (mg/g): NaCl - 105.0, KC1 - 120, KH2P04 - 310, Ca,(P04)* - 149, CaC03 - 210, MnS04 (anhydrous) - 0.20, KzAlz(S04)4 - 24 Hz0 - 0.09, MgS04 (anhydrous) - 90.0, FeP04 * 4 Hz0 - 14.7, CuS04 - 5 Hz0 - 0.39, NaF - 0.57, KI - 0.05. The following trace elements were also added (mg/kg diet): ZnClz - 15.0 and COClz - 6 Hz0 - 0.15. The vitamin mixture contained (mg/lOO g diet): thiamine - 0.8, riboflavin 0.8, pyridoxine -0.6, niacin - 5.0, calcium pantothenate - 4.0, inositol20.0, choline chloride - 200, folic acid - 0.4, cyanocobalamin - 2.0 pg, biotin - 20 pg, vitamin A - 1000 IU, vitamin D - 150 IU, e-tocopherol - 12 mg and menadione - 0.3 mg. The high fat cholesterol diet contained (g/100 g): dextrin - 50, casein - 20, coconut oil - 15, cholesterol - 5, salt mixture - 4, vitamin mixture - 1, cellulose - 4.5, sodium cholate - 0.5. The vitamin mixture and salt mixture had the same composition as given above. The animals were maintained on the respective diets for a period of 3 months. They were then starved overnight, stunned by a blow at the back of the neck and killed by decapitation. The tissues were quickly removed and placed in ice-cold containers for the various estimations. Determination of total ascorbic acid in the tissues was carried out by the dinitrophenylhydrazine method of Roe and Kuether [ 211. Lipid levels of the tissues Total cholesterol was determined by the method of Schoenheimer and Sperry [ 221, phospholipids by the method of Zilversmit and Davis [ 231 and triglycerides by the method of Van Handel and Zilversmit [24], with the modification that a Florisil column was used to remove phospholipids. Preparation of liver, kidney and spleen homogenates The liver was homogenised in 4 vols./g cold iso-osmotic
(0.25
M) sucrose
65
solution at 5°C in an all glass PotterElvehjem homogeniser. The kidney spleen were homogenised in 3 vols./g 0.15 M KC1 in the same way.
and
System for the study of biosynthesis of L-ascorbic acid from D-glucoronolactone The system used was the same as described by Mukherjee et al [25] and contained sodium phosphate buffer pH 7.4 (20 mM), D-glucuronolactone (10 mM), tissue homogenate equivalent to approximately 100 mg of tissue, sodium pyrophosphate (50 n-&I) and KCN (50 m&f). The total volume was 2.5 ml and the mixture was incubated at 37°C for 90 min. The ascorbic acid synthesized was determined titrimetrically using 2,6-dichlorophenol-indophenol [ 261. System for the study of catabolism of L-ascorbic acid The system used was the same as that described by Mukherjee et al [25] and contained dehydroascorbic acid (10 pmols, freshly prepared by Brz oxidation of ascorbic acid), tissue homogenate (equivalent to 100 mg of tissue), glutathione (0.3 pmol), Tris-maleate buffer pH 6.8 (200 pmols) and magnesium chloride (20 pmols). The final volume was 3.0 ml. The system was incubated at 37” for 15 min. The reaction was stopped by addition of 1 ml of 20% w/v metaphosphoric acid containing 2% w/v of SnClz . The remaining dehydro-ascorbic acid was rapidly reduced with HZ S and filtered. Excess of HZ S was removed by bubbling Nz. A aliquot (0.1 ml) was taken for the estimation of 2.3 diketogulonic acid formed according to the method of Kajawa et ai. [27]. A zero time control was also run. Results (1) Concentration of ascorbic acid in the tissues The results are given in Table 1. The concentration of ascorbic acid in the serum, liver, spleen and adrenals significantly decreased in the animals fed the atherogenic diet when compared with those fed the normal diet. The decrease in the concentration of ascorbic acid in the kidney was not, however, significant. (2) Relation between the concentration of ascorbic acid and the concentration of lipids in the tissues The results are given in Table 1. Except in the kidney, the decreased concentration of ascorbic acid in the tissues was associated with a significant increase in total cholesterol, phospholipids and triglycerides. (3) Ascorbic acid synthesis in the liver, spleen and kidney The results are given in Table 2. The synthesis of ascorbic acid from D-glucuronolactone was not significantly affected in the liver and kidney, but decreased significantly. in the spleen. (4) Catabolism of ascorbic acid in the liver, spleen and kidney The results are given in Table 2.
TABLE 1
44.8 f 1.16
45.0 f 0.93
20.8 * 0.82
402.0 f 12.00
Liver
Spleen
Kidney
Adrenals
501 f 12.1
288.4 f 6.65
270.5 zk8.8
355.6 f 13.3
73.5 f 2.44
Triglyceride (mg glycerol/ 100 g tissue) f SEM 6.5 f 0.18 780 f 23.8 680 f 12.66 80.8 f 2.01 1123 * 2.95 ____
Phosphollpid (mg/lOO g tissue) * SEM 136.5 f 4.21 1313 i: 40.1 847.2 k 21.84 2450.1 f 49.02 1224.0 ?J31.2 __ ___
Average of the values from the individual tissues of 6 rats. Group I has been compared with Group II. aP < 0.01. b 0.01 < P < 0.05. In alI other cases P > 0.05 ’ mg/lOO ml of serum.
2.4 f 0.055
Ch&SteK31
(mg/lOO g tissue) + SEM
g
Ascorbic acid
I
ACID AND LIPIDS IN TISSUES
(mg/100 tia¶le) ? SEM
Group
OF ASCORBIC
Serum c
Tissue
CONCENTRATION
206.0 f 4.4 a ___
18.2 + 0.54
34.5 f 1.24 a
26.5 ?r 0.98 a
1.40 f 0.051 =
(mg/lOO g tie) t SEM
Ascorbic acid
Group II
879 f 20.4 a
a
2080 + 56.2 a
3520 f 116.5
a
1565 f 41.5 a
552.1 * 14.41 442 + 10.83
2885 f 59.3 a
876.2 f 20.8 a a
150.1 f 3.11 b
201.5 f 4.65 a
Phospholipid (mg/lOO g tissue) f SEM
Cholesterol (mg/lOO g tissue) +_SEM
311.0 ?r 8.1 a
89.9 f. 3.91
620 t 81.5 a
1050 f 21.5 a
11.8 + 0.43 a
(mg glycerol/ 100 g tissue) f SEM
Triglyceride
67 TABLE 2 ASCORBIC
ACID SYNTHESIS
AND DEGRADATION
CHOLESTEROL
DIET
Tissue
Ascorbic acid synthesized (rg/h/g + SEM -_
Liver Spleen Kidney
IN RATS
protein)
FED A NORMAL
AND A HIGH FAT
Diketogulonic acid (&h/g * SEM __
protein) _______
Group I
Group II
Group I
Group II
2250 + 45.6 352 i- 7.06 285 + 10.0
2260 + 66.7 281 + 8.05 a 260 * 5.50
48.8 f 0.98 96.4 f 3.70 41.4 f 1.42
66.8 i 1.96 a 273.6 + 5.92 a 38.0 ? 0.91
Average of the valued from the individual tissue of 6 rats. Group I has been compared with Group II. a P < 0.01. In all other cases P > 0.05.
The catabolism of ascorbic acid as measured by the diketogulonic acid formed was significantly increased in the liver and spleen. But there was no significant alteration in the kidney. Discussion The decreased concentration of ascorbic acid found in the tissues of rats fed an atherogenic diet agrees, except in the case of the kidney, with result8 reported by Chevnyavskaya [18] in rabbit8 fed cholesterol. It does, however, not agree with Ginter’s [20] findings in rabbit8 and rats, where increased tissue levels were found. The concentration of ascorbic acid in the tissues is inversely related to that of lipids, in agreement with Chevnyavskaya [ 181. No report8 are available about the synthesis and catabolism of ascorbic acid in rats fed a high fat cholesterol diet, except for a report [19] that prolonged administration of cholesterol to guinea pigs significantly stimulated the oxidation of ascorbic acid to co*. The results now obtained indicate that the synthesis of ascorbic acid from Dglucuronolactone did not significantly differ in liver and kidney, but decreased in the spleen of rats fed a high fat cholesterol diet. On the other hand, the catabolism of ascorbic acid significantly increased in the liver and spleen, but not in the kidney. Thus, the decreased concentration of ascorbic acid in the spleen in rats fed the high fat cholesterol diet might have been due to its decreased synthesis and increased catabolism, while in the liver it might have been due to increased catabolism, synthesis being unaffected. The fact that the synthesis and catabolism of ascorbic acid are not affected in the kidney is consistent with it8 unaltered concentration in the tissues. The pathway for the synthesis catabolism of ascorbic acid is given below: The enzymes concerned with the Synthesis from D-glucuronolactone are Dglucuronolactone reductase and L-gulonolactone oxidase. The decreased synthesis of ascorbic acid in the spleen of rat8 fed the high fat high cholesterol diet indicates that the activity of these enzymes may be decreased in the spleen, but unaffected in the liver and kidney. The enzyme in-
68 Dghmuronolactone Dglucuronolactone I! 11 II
uronolact + onase D&uronic acid
reductase
4 + L-gulonolactone IE II II + G~onic acid
Lgulonolactone
I
oxidase
1 2-keto-L-gulonolactone
I I
1f- spontaneous + Ascorbic acid 1 dehydroascorbic
1
acid
+ dehydroascorbatsse
2 Lxylonl~
: 3 dlketogulonic acid
acid
L-lyxonic ’ acid
volved in the degradation of dehydroascorbic acid to 2,3_diketogulonic acid is dehydroascorbitase. The increased concentration of 2,3-diketogulonic acid in the liver and spleen in the rats fed the high fat cholesterol diet indicates increased activity of this enzyme. Activity of this enzyme is, moreover, presumably unaltered in the kidney. References 1 Sitaramayya. C. and Ali. T.. Experimental hypercholesterolemia and atherosclerosis, Indian J. Physiol. Pharmacol.. 6 (1962) 192. 2 Abramron. M.A., Mechanism of ascorbic acid action in alimentary cholesterol atherosclerosis in rabbits, Materialy. Nauch. Konf. PO problem am Fuakts. Vzalmootnosheniya Mezdu Raslichen Sistemami Organirma Y Norme i Palos Ivanova Sb. (1962) 638. 3 Volkova, K.G.. Effect of ascorbic acid on total lipids in the aortic wall in experimental cholesterol atherosclerosis, Ateroskleroz. Sb.. (1961) 187. 4 Fernandez. M.A.. Gimeno, Lacuara. J.L., Gimeno, A.L., Lema. B. and Malinow, M.R.. Effect of ascorbic acid on experimental atherosclerosis in chickens, Acta Physiol. Latinoam. 10 (1960) 168. 5 Chang, S.C., Effect of mechanical trauma, cortisone and vitamin C upon atherosclerosis in cholesterol fed rats, New Engl. Med. J., 8(8) (1965) 79. 6 Sokoloff, B., Hori, M.. Sadhof, C.. McConnell, B. and Imai. T., Effect of ascorbic acid on certain fat metabolism factors in animals and man, J. Nutr.. 91 (1967) 107. 7 Rzakulleva. D.M.. Effect of ascorbic acid on the development of atherosclerosis, Izv. Akad. Nauk. Azerb. SSR. Ser Biol. Nauk. No. 2 (1969) 110. 8 Novitskil. A.A., Ivanov, A.I. and Reshetnev. V.G.. Changes of intensity of cholesterol biosynthesis under the effect of ascorbic acid at early stages of experimental atherosclerosis, Patol. Fiaiol. Eksp. Ter.. 13 (1969) 59. 9 Ginter. E., Ondreicka, R.. Bobek, P. and Simko. V.. Influence of chronic vit. C deficiency on fatty acid composition of blood serum, liver triglycerides and cholesterol esters in guinea pigs, J. Nutr. 99 (1969) 261. 10 Giiter. E., Babala, J. and Cerven, J.. Effect of chronic hypovitaminosis C on the metabolism of cholesterol and atherogenesls ln guinea pigs, J. Atheroscler. Res.. 10 (1969) 341. 11 Ginter. E. and Nemco, R.. Metabolism of [1-14C]acetate in guinea pids with chronic vitamin C hyposaturation, J. Atheroscl. Res., 10 (1969) 273. 12 Novitskii. A.A.. Correlation of the metabolism of cholesterol and ascorbic acid during prolonged hypercholesterolemea. Tr. Kuibyshev. Med. Inst., 56 (1969) 122. 13 Novitskii. A.A., Effect of ascorbic acid on the biosynthesis of cholesterol in the early stages of experimental atherosclerosis, Kardiologiya, 10 (1970) 118. 14 Ginter, E., Babala, J. and Polonyova, E., Vitamin C and lipid metabolism in rabbits fed an atherogenic diet, Biologia. 25 (1970) 579. 15 Glnter, E. and Babala, J., Effect of high intraperitoneal doses of vitamin C in guinea pigs fed atherogenie diet, Biologia. 26 (1971) 449. 16 Novitskli, A.A.. Influence of vitamin C on cholesterol netabolism in liver in experimental atherosclerosis, CoretVasa, 11 (1969) 302. 17 FWinami. T., Okado, K.. Senda. K., Sugimura, M. and Klshlkawa, M., Experimental atherosclerosis
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